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The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and
1Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-5222, USA.
Abstract:
A variety of pathogenic bacteria use type III secretion pathways to translocate virulence proteins into host eukaryotic cells. YopE is an important virulence factor that is translocated into mammalian cells via a plasmid-encoded type III system in Yersinia spp. YopE action in mammalian cells promotes the disruption of actin filaments, cell rounding and blockage of phagocytosis. It was reported recently that two proteins with sequence similarity to YopE, SptP of Salmonella typhimurium and ExoS of Pseudomonas aeruginosa, function as GTPase-activating proteins (GAPs) for Rho GTPases. YopE contains an 'arginine finger' motif that is present in SptP, ExoS and other Rho GAPs and is essential for catalysis by this class of proteins. We show here that a GST-YopE fusion protein stimulated in vitro GTP hydrolysis by the Rho family members Cdc42, RhoA and Rac1, but not by Ras. Conversion of the essential arginine in the arginine finger motif to alanine (R144A) eliminated the in vitro GAP activity of GST-YopE. Infection assays carried out with a Yersinia pseudotuberculosis strain producing YopER144A demonstrated that GAP function was essential for the disruption of actin filaments, cell rounding and inhibition of phagocytosis by YopE in HeLa cells. Furthermore, the GAP function of YopE was important for Y. pseudotuberculosis pathogenesis in a mouse infection assay. Transfection of HeLa cells with a vector that produces a constitutively active form of RhoA (RhoA-V14) prevented the disruption of actin filaments and cell rounding by YopE. Production of an activated form of Rac1 (Rac1-V12), but not RhoA-V14, in HeLa cells interfered with YopE antiphagocytic activity. These results demonstrate that YopE functions as a RhoGAP to downregulate multiple Rho GTPases, leading to the disruption of actin filaments and inhibition of bacterial uptake into host cells.
Insights
Yersinia bacteria use YopE to disrupt host cell actin and block phagocytosis by acting as a GTPase-activating protein (GAP) for Rho GTPases. This bacterial virulence mechanism is crucial for Yersinia pseudotuberculosis pathogenesis in mammals.
Area of Science:
- Microbiology
- Cell Biology
- Bacterial Pathogenesis
Background:
- Pathogenic bacteria employ type III secretion systems to deliver virulence factors into host cells.
- Yersinia spp. utilize YopE, a key virulence factor, to disrupt host cell actin cytoskeleton and inhibit phagocytosis.
- Homologs of YopE, such as SptP and ExoS, function as GTPase-activating proteins (GAPs) for Rho GTPases.
Purpose of the Study:
- To investigate the GTPase-activating protein (GAP) activity of Yersinia YopE.
- To determine the role of YopE's GAP function in Yersinia pathogenesis and host cell manipulation.
- To elucidate the specific Rho GTPases targeted by YopE.
Main Methods:
- Biochemical assays to assess GTP hydrolysis by YopE fusion proteins on Rho GTPases.
- Site-directed mutagenesis to investigate the importance of the YopE arginine finger motif.
- Infection assays using Yersinia strains with wild-type and mutant YopE in cell culture (HeLa cells).
- Mouse infection models to evaluate the in vivo role of YopE's GAP activity.
- Cell-based assays involving transfection with constitutively active Rho GTPases.
Main Results:
- GST-YopE demonstrated in vitro GTPase-activating protein (GAP) activity towards Cdc42, RhoA, and Rac1, but not Ras.
- Mutation of the conserved arginine in the YopE arginine finger motif abolished GAP activity.
- YopE's GAP function was essential for actin filament disruption, cell rounding, and phagocytosis inhibition in HeLa cells infected with Yersinia pseudotuberculosis.
- The GAP activity of YopE was critical for Y. pseudotuberculosis pathogenesis in a mouse model.
- Expression of constitutively active RhoA (RhoA-V14) in HeLa cells blocked YopE-mediated actin disruption and cell rounding.
- Activated Rac1 (Rac1-V12), but not RhoA-V14, interfered with YopE's antiphagocytic effects.
Conclusions:
- YopE functions as a Rho GTPase-activating protein (GAP).
- YopE downregulates multiple Rho GTPases, including Cdc42, RhoA, and Rac1.
- This downregulation leads to actin cytoskeleton disruption and inhibition of bacterial uptake by host cells.
- YopE's GAP activity is a critical virulence mechanism for Yersinia pseudotuberculosis infection.